With the recent completion of sequencing of the human genome and continuing development of more rapid and sensitive protein analytical methods requiring sample volumes of a few uL or less, such as mass spectrometry, surface plasmon resonance, x ray crystallography, Enzyme Linked Immuno Sorbent Assay (ELISA) and nuclear magnetic resonance, there is a growing need for more cost-effective tools for purification and analysis of very small amounts of proteins.
Microporous polymeric membranes and nonwoven polymeric and inorganic filtration media are widely used in biological research to capture particulates and cells or cell fragments from fluids. The filters may be selected or chemically modified to directly capture dissolved molecules by ionic, hydrophobic, hydrogen bonding, or affinity interactions with the filter itself. Neutral inert filters are commonly used to retain packed beds of porous or nonporous chromatographic beads having such interactions, and to retain beds of size-exclusion beads having pores of a size useful to separate macromolecules larger than the pores from molecules able to enter the pores.
Similar to reactive membranes, porous monoliths may be prepared which have porous (e.g. U.S. Pat. Nos. 6,048,457; 6,200,474; 6,653,201) or nonporous (e.g. U.S. Pat. App. No. 20,020,164,818) beads fused or liked together to form a self-supporting shaped article commonly contained in a conical pipette tip which thereby need not incorporate a support filter. This construction has the advantage of eliminating need for a bed supporting means, but adds the cost of multiple process steps on each device, and can introduce new sources of variability between devices resulting from the added operations, as compared to beds physically packed from beads which are all prepared as a uniform batch.
Fluids are commonly moved through such devices by application of controlled difference in either air or in hydrostatic fluid pressure. Single devices and arrays of 8 or 12 discrete devices are commonly formed in molded plastic pipette tips, permitting sequential filling by aspiration and emptying by extension of the pipettor piston to create air pressure in each tip device. Arrays of such devices are formed in multiwell filter plates having 96, 384 and larger numbers of wells each having a filter disc forming the bottom surface of the well. These plates are filled and handled by robotic pipettors and filtration is easily obtained using a vacuum manifold, which may contain a collection plate having mating wells to receive each filtrate.
Alternatively, single devices or filterplates are placed in collection tubes or multiwell plates, respectively, and spun in a centrifuge to generate hydrostatic pressure to cause filtration and percolation through a particulate bed.
A variety of methods are used to manufacture single and multiwell filters for these applications. Individual disks may be punched or die cut and deposited into the base of pre-molded sleeves (U.S. Pat. Nos. 4,774,058; 6,482,362 B1). This is a multi-step process and leaks may develop if the loose filters are dislodged in shipment or use.
Disks may be welded or bonded to cover the tips of pre-molded sleeves or be crush captured between mating upper and lower sleeve segments (U.S. Pat. Nos. 4,902,481 and 5,833,927; U.S. Pat. Apps. 20,040,072,375 and 20,040,142,488). This construction requires multiple parts and steps, and is difficult to practice to form beds having volumes of 10 uL or less and low dead volume.
Segments of a single sheet of filter may be sealed and isolated by the process of ultrasonic welding together of upper and lower trays having mating chambers and apertures. Prevention of cross contamination between wells requires having the mating chambers circumscribed by means to cut through the sheet around each chamber as part of the weld (U.S. Pat. Nos. 4,948,442 and 5,047,215). This process would not be practical to produce individual beds having volumes of 10 uL or less with low dead volume.
A lower plate or part may be molded, having at least one cavity to receive a punched disk and to deliver filtrate through an aperture, which is then captured and sealed by insert molding a second part forming a sample well or chamber above it which bonds to the disk and to the first part (U.S. Pat. No. 6,391,241). This process requires three separate operations.
Many other methods are known in the art for fabrication of devices containing filters by insert molding (e.g. U.S. Pat. Nos. 4,601,820; 4,812,216; 5,429,742; and 5,922,200). In each case a piece of membrane or filter is first pre-cut, one or more supporting members are optionally pre-molded, the precut filter and optional pre-molded support are inserted in a mold cavity, and a thermoplastic housing is thereby formed around the filter. This known method of insert molding imposes the requirement of a high precision punch and die set to cleanly cut difficult materials such as nonwoven polypropylenes, which tend to shred and resist punching. Even with a high precision die set, considerable care is needed to consistently place and seal a punched disk, which may be less than 0.03 inch in diameter, into a micro-volume insert mold chamber.
Accordingly, there remains a need for improved methods and devices for preparing and separating biological analytes.